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Annals of Surgical Oncology 10:582-587 (2003)
© 2003 Society of Surgical Oncology

NEW APPROACHES TO THE TREATMENT OF HEPATIC MALIGNANCIES

Platelet-Derived Endothelial Cell Growth Factor Mediates Rho-Associated Coiled-Coil Domain Kinase Messenger RNA Expression and Promotes Cell Motility

Keiji Yoshinaga, PhD, MD, Hiroshi Inoue, PhD, MD, Fumiaki Tanaka, PhD, MD, Koshi Mimori, PhD, MD, Tohru Utsunomiya, PhD, MD and Masaki Mori, PhD, MD

From the Department of Molecular and Surgical Oncology, Medical Institute of Bioregulation, Kyushu University, Beppu, Japan.

Correspondence: Address correspondence and reprint requests to: Masaki Mori, MD, PhD, Department of Molecular and Surgical Oncology, Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumibaru, Beppu, 874-0838, Japan; Fax: 81-977-27-1651; E-mail: mmori{at}beppu kyushu-u.ac.jp.

ABSTRACT

Background: Platelet-derived endothelial cell growth factor (PD-ECGF), whose expression is increased in several cancers, is an endothelial cell mitogen and has chemotactic activity in vitro and angiogenic activity in vivo. Tumors with high PD-ECGF expression tend to have frequent lymph node metastasis and are associated with poor outcome.

Methods: We screened genes transduced by PD-ECGF transfection to the colon cancer cell line DLD-1 by using a complementary DNA microarray. Cell motility was evaluated by in vitro migration assay. Actin fiber polymerization was visualized by immunofluorescent detection of phalloidin.

Results: Rho-associated coiled-coil domain kinase (ROCK1) was found to be significantly overexpressed in PD-ECGF transfectants compared with mock cells. PD-ECGF transfectants showed higher cell motility than mock cells. The parental cell, DLD-1, with recombinant PD-ECGF showed higher cell motility than that without recombinant PD-ECGF, in which motility was blocked by the neutralizing antibody of PD-ECGF or Y-27632, a specific inhibitor of ROCK1. Moreover, the actin fiber polymerization, which is a marker of activation of ROCK1, was higher in PD-ECGF transfectants than in mock cells.

Conclusions: PD-ECGF expression may be associated with cancer cell migration via activation of ROCK1. This may explain one mechanism by which tumors with high expression of PD-ECGF show aggressive behavior.

Key Words: PD-ECGF • ROCK1 • Cell motility • cDNA microarray

Platelet-derived endothelial cell growth factor (PD-ECGF) is, as is thymidine phosphorylase, a member of the pyrimidine nucleoside phosphorylase family and is essential for DNA synthesis and limiting cell growth. The overexpression of PD-ECGF has been reported in solid carcinoma and is associated with lymph node metastasis, angiogenesis, and poor prognosis.1–3 Moreover, the transfectant of PD-ECGF in human KB epidermoid carcinoma cells showed high-grade liver metastasis.4 PD-ECGF is also a metabolic factor of thymidine as thymidine phosphorylase. The degradation products, thymine and 2-deoxy-D-ribose, have angiogenic and antiapoptotic effects that are associated with tumor aggressiveness and poor survival.5 Thus, to clarify the role of PD-ECGF in metastasis, we compared the gene expression profiles of PD-ECGF transfectants with those of mock cells by complementary DNA (cDNA) microarray. Consequently, we detected Rho-associated coiled-coil domain kinase (ROCK1), which was the only overexpressed gene among all the subclones, and keratin 18, which was the only suppressed gene among all the subclones. In this study, we focused on the expression and activation of ROCK1 by PD-ECGF in the mechanism of aggressive behavior of tumors with high PD-ECGF expression.

METHODS

Cell Culture
DLD-1 mock cells (Va) and transfectants of PD-ECGF (Ta, Te, and Tc), provided by Dr. Tanaka (Nippon Roche Co., Tokyo, Japan), were maintained in RPMI 1640 culture medium (Nissui Pharmaceutical, Tokyo, Japan) with 10% fetal bovine serum (Equitech-Bio, Inc., Kerrville, TX) in 95% air and 5% CO2 atmosphere at 37°C. Human umbilical vessel endothelial cells (HUVEC) were maintained in MCDB131 culture medium (Life Technologies, Inc., Rockville, MD) with 10% fetal bovine serum. Twenty-four hours after the first passage, the culture medium of DLD-1 was replaced with or without 2.0 to 200 ng/mL of human recombinant PD-ECGF (R&D Systems, Minneapolis, MN) for 36 hours of exposure. Twenty-four hours after the first passage, the culture medium of HUVEC was replaced with or without human recombinant PD-ECGF (3.0–300 ng/mL) for 36 hours of exposure.

Proliferation Assay
Cells were seeded in triplicate in 6-cm dishes at a concentration of 1 x 105 cells per dish. Cells were collected daily for 5 days, and the number of viable cells, assessed by trypan blue exclusion, was counted in a hemocytometer.

Western Blot Analysis
DLD-1 mock cells and transfectants were lysed in 1 mL of 1x sample buffer consisting of 50 mM of Tris-HCl (pH 7.5), .5 M of NaCl, and 10% glycerol. After centrifugation, protein (30 µg) was separated on a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel and electroblotted onto a nylon membrane (Millipore, Bedford, MA). For immunoblotting, 1000x anti–PD-ECGF antibody was used, as described previously.6 Secondary horseradish peroxidase–conjugated antibodies (Amersham Pharmacia Biotech, Piscataway, NJ) were used and were visualized with an enhanced chemiluminescence system (Amersham Pharmacia Biotech). The expression of PD-ECGF messenger RNA (mRNA) and protein was ensured (Fig. 1a).



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  		FIG. 1. (a) The expression of platelet-derived endothelial cell growth factor (PD-ECGF) and the control gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), in mock cells (Va) and transfectants (Ta, Te, Tc) is demonstrated by reverse transcription-polymerase chain reaction analysis (upper, middle) and Western blot analysis (bottom). Although the expression level of G3PDH is almost the same among the three transfectants, that of PD-ECGF is much different; Tc is the highest, followed by Te and Ta. (b) Cell growth curves demonstrate that the cell count is not significantly different among the cells; Va, Ta, Te, and Tc, n = 6. NS, not significant.

 
Complementary DNA Microarray Analysis
The cDNA microarray Human Cancer Chip version 2.0 (TaKaRa Biochemicals, Tokyo, Japan) was used. The analysis was performed as follows. For competitive hybridization, Cy5-labeled or Cy3/deoxyuridine triphosphate–labeled (Amersham Pharmacia Biotech) cDNAs were derived from 15 µg of total cellular RNA of DLD-1 mock (Va) and PD-ECGF transfectants (Ta, Te, Tc) by an RNA labeling kit (TaKaRa Biochemicals). A microarray was hybridized with probes. After overnight hybridization at 65°C, the slide was washed in 2x standard saline citrate (SSC) and .5% SDS for 30 minutes at 55°C twice, in 2x SSC and .5% SDS for 5 minutes at 65°C, and in .05x SSC and .5% SDS for 5 minutes at room temperature. The slides then were scanned with GMS418 (Molecular Dynamics, Sunnyvale, CA), with sample intensities for Cy3 and Cy5 collected separately. The intensity of each hybridization signal was evaluated photometrically with the Imagene computer program (BioDiscovery, Marina Del Rey, CA) and normalized to the averaged signals of a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. The Cy3:Cy5 ratio for each sample was calculated by averaging spots. The cutoff value for each expression level was calculated according to the background fluctuation. We used the expression level (>100) where the fluctuation is less than a critical value (1.0) because the other genes (those that have a low expression) are embedded in the background fluctuation. We defined the upregulated and downregulated genes in PD-ECGF transfectants as a Cy3:Cy5 signal ratio >2.0 and <.50, respectively.

Reverse Transcription-Polymerase Chain Reaction
Complementary DNAs were synthesized from 2.5 µg of total RNA in a 25-µL reaction mixture as described previously.7 The products were loaded on 1.2% agarose gels with the concentration of 400 ng/mL of ethidium bromide and electrophoresed. The primer sequences of PD-ECGF and ROCK1 were as follows: up, 5'-GCCCCTCCACGAGTTTCTTACT; down, 5'-GGGGGTGTGGGTGACAAGGTC; and up, 5'-GCCGTCCGAAAGATGCTGTGG; down, 5'-CGTTTGATTTCTTCTACACCA, respectively. Both polymerase chain reaction (PCR) amplifications were performed for 22 cycles under the following conditions: (1) denaturing at 95°C for 1 minute, (2) annealing at 54°C for 1 minute, and (3) polymerization at 72°C for 1 minute.

Migration Assay
Confluent serum-deprived monolayer cultures of DLD-1 cells (mock and transfectants) were incubated with or without human recombinant PD-ECGF and/or mouse anti–PD-ECGF neutralizing antibody or ROCK1 inhibitor, Y-27632, in 8-µm pored culture membrane inserts (Becton Dickinson Immunocytometry System, Mountain View, CA). Y-27632 was provided by Mitsubishi Pharma Co. (Osaka, Japan). After 36 hours of incubation, cells on the upper side of the insert chamber were wiped off. The migrating cells on the lower side of the insert were observed with a microscope. The original magnification was x100.

Immunofluorescent Detection
Cells were seeded on coverslips (Becton Dickinson Immunocytometry System), incubated with various agents in serum-free medium for 36 hours, stained for F-actin by fixing the cells in 4% paraformaldehyde followed by incubation with rhodamine-conjugated phalloidin (Molecular Probes Inc., Eugene, OR), and visualized on a Carl Zeiss (Oberkochen, Germany) Axovert fluorescence microscope.

Statistical Analysis
Differences with a P value of <.05 with Student’s two-tailed t-test were considered significant.

RESULTS

The Expression of PD-ECGF in Transfectants and Mock Cells
Western blot and reverse transcription-PCR analyses revealed the expression of PD-ECGF in DLD-1 subclones of mock cells (Va) and transfectants (Ta, Te, and Tc). Tc showed the highest expression of PD-ECGF, followed by Te and Ta. However, Va showed no expression of PD-ECGF (Fig. 1a). Moreover, a proliferation assay showed no significant difference of growth rate among all subclones (Fig. 1b).

The Expression of ROCK1 mRNA Mediated by PD-ECGF
We performed cDNA microarray analysis to obtain global mRNA expression profiles of all PD-ECGF transfectants and mock cells (data not shown), and we identified ROCK1 as an overexpressed gene when compared with mock cells. The expression ratios between subclones of mock Va and transfectants Ta, Te, and Tc were 2.62, 5.89, and 2.33, respectively. It is interesting to note that Te, with the second highest expression of PD-ECGF, showed the highest expression of ROCK1 (Fig. 2a). We investigated the expression of ROCK1 in another cell line, HUVEC, which is the cell line without PD-ECGF mRNA expression after coculture with human recombinant PD-ECGF. At a low dose of recombinant PD-ECGF (3–30 ng/mL), ROCK1 mRNA expression was higher than that of the control. PD-ECGF 30 ng/mL induced the highest ROCK1 mRNA expression in this study. However, the highest dose (300 ng/mL) of PD-ECGF suppressed the expression of ROCK1 (Fig. 2b).



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  		FIG. 2. (a) Messenger RNA (mRNA) expression of Rho-associated coiled-coil domain kinase (ROCK1) in mock (Va) and platelet-derived endothelial cell growth factor (PD-ECGF) transfectants (Ta, Te, Tc). The expression is the highest in Te, followed by (b) mRNA expression of ROCK1 in human umbilical vessel endothelial cells (HUVEC) with or without recombinant PD-ECGF. The expression is highest with the middle-dose PD-ECGF (30 ng/mL), followed by the low dose (3.0 ng/mL). Note that it is suppressed at high-dose PD-ECGF (300 ng/mL). G3PDH, glyceraldehyde-3-phosphate dehydrogenase.

 
Increased Cell Motility by PD-ECGF
It is known that ROCK1, which is downstream of Rho A, the so-called rac/rho signaling, contributes to cell motility and is inhibited by C38 and toxin A,9 which are specific inhibitors of Rho A and Y-27632, themselves specific inhibitors of ROCK1.10 Therefore, we studied the cell motility in the transfectant (Te) with the highest expression of ROCK1 and in mock cells (Va) in a migration assay with stimulation by fetal bovine serum. The subclone of PD-ECGF transfectant, Te, showed higher migration activity than mock cells. To examine whether these effects were caused by PD-ECGF and ROCK1, the migration assay was performed under the same conditions with or without recombinant PD-ECGF, anti–PD-ECGF neutralization antibody, or Y-27632 in DLD-1 (Fig. 3). The activity was increased in the presence of 20 ng/mL of recombinant PD-ECGF. Neither a lower (2.0 ng/mL) nor higher (200 ng/mL) dose of PD-ECGF increased cell motility (data not shown). This activation was inhibited by PD-ECGF neutralizing antibody and Y-27632.



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  		FIG. 3. Left panel: average number of migrating cells in three experiments of mock (Va) and transfectant (Te) cells. *P < .05. Right panel: number of migrating DLD-1 cells in several conditions. PD-ECGF, platelet-derived endothelial cell growth factor; hr-PD-ECGF, human recombinant platelet-derived endothelial cell growth factor; Ab, antibody.

 
Activation of ROCK1 by PD-ECGF
We assessed the degree of actin fiber formation after activation of ROCK1. The mock cells, Va, did not form stress fibers and exhibited a little polymerization (Fig. 4a). A subclone of PD-ECGF transfectant Te showed a higher degree of actin fiber polymerization than that of Va (Fig. 4b). In the parent cells, DLD-1, culture with 20 ng/mL of human recombinant PD-ECGF showed greater actin fiber polymerization than under the recombinant PD-ECGF–free condition (Fig. 4c,d). This effect was inhibited by PD-ECGF neutralizing antibody and Y-27632 (Fig. 4f,g).



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  		FIG. 4. Actin fiber staining by rhodamine-conjugated phalloidin. White arrows indicate actin polymerization. (a) Actin polymerization in mock cells (Va). (b) Increase of actin polymerization in transfectant cells (Te). (c) Slight actin polymerization is recognized in DLD-1. (d) Actin polymerization is increased in DLD-1 with recombinant platelet-derived endothelial cell growth factor (PD-ECGF; 20 ng/mL). (e) Actin polymerization in DLD-1 is remarkably inhibited with PD-ECGF neutralizing antibody (20 µg/mL). (f) Actin polymerization is also inhibited with Y-27632, a specific inhibitor of Rho-associated coiled-coil domain kinase (ROCK1; 10 µM) in DLD-1.

 
DISCUSSION

The high expression of PD-ECGF results in enhanced angiogenesis, high-grade lymph node metastasis, vascular permeation, liver metastasis, and poor prognosis. The expression of PD-ECGF is correlated with that of vascular endothelial cell growth factor and microvessel count.3,11 PD-ECGF increases the growth of endothelial cells, such as HUVEC.12 However, the mechanisms of lymph node metastasis, vascular permeation, and liver metastasis mediated by PD-ECGF are still unclear. To clarify the effects of PD-ECGF on carcinoma cells, we used stable transfectants of PD-ECGF and compared the mock cells and transfectants.

Only the ROCK1 gene related to cell motility was more highly expressed in all subclones of transfectants (Ta, Te, and Tc) than mock cells (Va). There are several reports that ROCK1 is activated by lysophosphatidic acid (LPA) and transformed growth factor-ß and increases cell motility in vitro.13,14 However, there have been no reports on whether mRNA expression of ROCK1 is mediated by LPA or transformed growth factor-ß. The kind of signaling that mediates mRNA expression of ROCK1 is still unknown. We demonstrated that transfectant Te with an appropriate expression level of PD-ECGF showed the highest expression of ROCK1. Moreover, HUVEC with 30 ng/mL of PD-ECGF increased ROCK1 expression compared with control and/or a lower dose (3.0 ng/mL). However, an excess dose (300 ng/mL) of PD-ECGF suppressed the expression of ROCK1. These results indicate that an appropriate dose of PD-ECGF mediates mRNA expression of ROCK1, and this may be one of the mechanisms by which excess doses of LPA and overexpression of Rho A suppress cell motility.13,15

We found that transfectant (Te) exhibited greater cell motility than mock cells (Va). A proliferation assay of these subclones showed no significant difference of growth rate. Thus, cell migration is not related to cell growth. DLD-1-cultured 20 ng/mL of recombinant PD-ECGF showed greater motility than DLD-1 without PD-ECGF, and this effect was inhibited by PD-ECGF neutralizing antibody and Y-27632. These results indicate that PD-ECGF increases cell motility via the activation of ROCK1. It is interesting to note that Itoh et al.10 reported that the activation of ROCK1 results in high-grade peritoneal dissemination in a mouse model.

ROCK1 increases cell motility via activation of the actomyosin cascade.16 ROCK1 regulates actin stress fiber formation, actin polymerization, and integrin activation. Transient activation of ROCK1 causes stress fiber formation, whereas chronic activation causes actin fiber polymerization.16 We observed actin fiber polymerization in transfectant (Te), mock cells (Va), and DLD-1 with or without recombinant PD-ECGF. We showed that ROCK1 in DLD-1 is activated by recombinant PD-ECGF and that polymerization is inhibited by PD-ECGF neutralizing antibody and Y-27632. Our findings suggest that PD-ECGF mediates the mRNA expression of ROCK1 and activates ROCK1. Thus, PD-ECGF may contribute to high-grade metastasis in vivo by mediating the expression of ROCK1 and its activation. The signaling of PD-ECGF as a growth factor and the mechanism by which PD-ECGF mediates ROCK1 are still unknown.

Contrary to contributing to poor prognosis, high expression of PD-ECGF enhances the effectiveness of anticancer drugs—for example, 5'-deoxy-5-fluorouridine (5'-dFUrd) and capecitabine (N4-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine)17–19—because 5-fluorouracil is converted to 5'-dFUrd by PD-ECGF.17,18 Capecitabine is converted to 5-fluorouracil by three enzymes located in the liver and tumors; the final step is the conversion of 5'-dFUrd to 5-fluorouracil by PD-ECGF in tumors.19 Thus, the expression of PD-ECGF is a double-edged sword in solid tumors in colorectal, gastric, breast, and esophageal cancers. Preventing metastasis without reducing the effects of anticancer drugs is important in solid tumors with high expression of PD-ECGF. Our findings show the possibility of preventing cell motility of cancer cells with high expression of PD-ECGF by inhibiting ROCK1 activation. Moreover, it is necessary to study whether cancer cells with high expression of PD-ECGF are more susceptible to anticancer drugs that inhibit ROCK1 activation.

Acknowledgments

The acknowledgments are available online at www.annalssurgicaloncology.org.

The authors thank Mitsubishi Pharma Co. (Osaka, Japan) for providing Y-27632 and Dr. Tanaka (Nippon Roche Co., Tokyo, Japan) for providing DLD-1 mock cells (Va) and PD-ECGF transfectant cells (Ta, Te, and Tc). The authors also thank Tomomi Murakami, Junko Miyake, Kazue Ogata, and Toshiko Shimooka for excellent technical assistance.

Footnotes

We screened genes transduced by platelet-derived endothelial cell growth factor (PD-ECGF) transfection to the colon cancer cell line DLD-1 by using complementary DNA microarray and found that Rho-associated coiled-coil domain kinase (ROCK1) was significantly overexpressed in PD-ECGF transfectants compared with mock cells. PD-ECGF transfectant showed higher cell motility than mock cells via activating ROCK1. This may explain one mechanism by which tumors with high expression of PD-ECGF show aggressive behavior.

Received for publication August 27, 2002. Accepted for publication January 29, 2003.

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